The invention relates to methods for quenching electrophiles.
The transmission of viral diseases (e.g., hepatitis A, B, and C infections, acquired immunodeficiency syndrome, and cytomegalovirus infection) by blood or blood products is a significant problem in medicine. Other biological compositions, such as mammalian and hybridoma cell lines, products of cell lines, milk, colostrum, and sperm, can also contain infectious viruses. Screening donor biological compositions for viral markers can help reduce the transmission of viruses to recipients, but many screening methods are directed to only a few discrete viruses, and are therefore incomplete, and may also be less than 100% sensitive. It is therefore important to inactivate viruses contained in donor blood, blood products, or other biological compositions.
A number of agents that are capable of inactivating viruses in blood have been developed. For example, ethyleneimine monomer and ethyleneimine oligomers are very effective viral inactivating agents. Methods for producing and using ethyleneimine oligomers for inactivating viruses in biological compositions are generally described in U.S. Ser. No. 08/835,446 (filed Apr. 8, 1997), U.S. Ser. No. 08/521,245 (filed Aug. 29, 1995), U.S. Ser. No. 08/855,378 (filed May 13, 1997), U.S. Ser. No. 09/005,606 (filed Jan. 12, 1998), and U.S. Ser. No. 09/005,719 (filed Jan. 12, 1998), which are hereby incorporated by reference. Ethyleneimine oligomers are themselves chemically active, and must therefore be rendered non-reactive before a product, such as blood, is used clinically. Typically, a viral inactivating compound, such as ethyleneimine dimer, is added to a biological composition to inactivate infectious viruses that might be present in the composition. A quenching agent is then added to inactivate the ethyleneimine dimer that remains after viral inactivation has taken place. The end result is a biological composition that is relatively free of infectious viruses, but that is contaminated with quenched inactivating agent and with quenching agent.
In one aspect, the invention features a method of inactivating a contaminant, such as a virus, of a biological composition; the method includes the steps of: (a) contacting the biological composition with an inactivating agent including an aziridino moiety, such as ethyleneimine, an oligomer of ethyleneimine, or a haloderivative salt of either ethyleneimine or an oligomer of ethyleneimine, where a portion of the agent reacts with and inactivates the contaminant, and a portion of the agent remains unreacted; (b) contacting the product of step (a) with a lipophilic quenching agent including at least one quenching moiety, under conditions and for a time sufficient to allow the unreacted agent to bond covalently to the quenching moiety; and (c) separating the lipophilic quenching agent and the quenched inactivating agent from the biological composition.
A preferred quenching moiety includes a nucleophilic moiety, such as a thiosulfate or thiophosphate moiety; the thiophosphate moiety may be part of an internucleotide linkage of an oligonucleotide sequence.
The inactivating agent may be, for example, ethyleneimine, an oligomer of ethyleneimine, or a haloderivative salt of either ethyleneimine or an oligomer of ethyleneimine. The biological composition may be selected from the group consisting of whole mammalian blood, purified or partially purified blood proteins, blood cell proteins, milk, saliva, blood plasma, platelet-rich plasma, a plasma concentrate, a precipitate from any fractionation of plasma, a supernatant from any fractionation of plasma, a serum, a cryoprecipitate, a cryosupernatant, a cell lysate, a mammalian cell culture, a mammalian culture supernatant, a placental extract, a product of fermentation, a platelet concentrate, a leukocyte concentrate, semen, red blood cells, and a recombinant protein-containing composition produced in a transgenic mammal. Preferably, the biological composition is whole human blood or human blood plasma. The contaminant may be a virus.
In a second aspect, the invention features a method of quenching an electrophile; the method includes contacting the electrophile with a composition including at least one thiosulfate or thiophosphate moiety attached to a second lipophilic moiety, under conditions and for a time sufficient to allow the electrophile to bond covalently to the thiosulfate or thiophosphate moiety. In preferred methods, a plurality of the thiosulfate or thiophosphate moieties are substituted with at least one C1-40 saturated or unsaturated hydrocarbon skeleton that is unsubstituted or has between 1 and 4, inclusive, substituents, independently selected from the group consisting of hydroxyl, amino, cyano, and azido.
Preferably, the electrophile includes an aziridino moiety or a haloderivative salt. For example, the electrophile may be ethyleneimine or an oligomer of ethyleneimine.
In a third aspect, the invention features a method of removing a viral inactivating agent from a biological composition; the method includes the steps of: (a) contacting the inactivating agent with a quenching agent that is coupled to a lipophilic moiety selected from the list consisting of linear, branched, or cyclic saturated or unsaturated hydrocarbons or esters with one to forty carbons, benzyl groups, or polycyclic aromatic groups, all of which may contain hydroxyl, amino, cyano, or azido substituents; and (b) removing the inactivating agent, the quenching agent, and the lipophilic moiety from the biological composition. Preferably, step (a) includes contacting the inactivating agent with the quenching agent under conditions and for a time sufficient to allow covalent bonds to form between the inactivating agent and the quenching agent. A preferred quenching agent includes a nucleophilic moiety, such as a thiosulfate or thiophosphate moiety.
In a fourth aspect, the invention features a compound comprising (a) a lipophilic moiety; and (b) a thiosulfate or thiophosphate moiety. Preferably, the lipophilic moiety is selected from the list consisting of linear, branched, or cyclic saturated or unsaturated hydrocarbons or esters with one to forty carbons, benzyl groups, or polycyclic aromatic groups, all of which may contain hydroxyl, amino, cyano, or azido substituents. The compound may further include a reporter moiety, such as UV adsorbing or fluorescent groups. The thiophosphate moiety may be part of an internucleotide linkage of an oligonucleotide sequence.
By xe2x80x9cquenching moietyxe2x80x9d or xe2x80x9cquenching agentxe2x80x9d is meant a thiophosphate or a thiosulfate, or a compound containing a thiophosphate or a thiosulfate moiety that, when contacted with an electrophile, such as an ethyleneimine oligomer, is capable of rendering the contacted electrophile non-reactive.
By xe2x80x9cbiological compositionxe2x80x9d is meant a composition containing cells or a composition containing one or more biological molecules, or a composition containing both cells and one or more biological molecules. Cell-containing compositions include, for example, mammalian blood, red cell concentrates, platelet concentrates, leukocyte concentrates, blood plasma, platelet-rich plasma, semen, placental extracts, mammalian cell culture or culture medium, products of fermentation, and ascites fluid. Biological compositions may also be cell-free, and contain at least one biological molecule. By xe2x80x9cbiological moleculexe2x80x9d is meant any class of organic molecule normally found in living organisms including, for example, nucleic acids, polypeptides, post-translationally modified proteins (e.g., glycoproteins), polysaccharides, and lipids. Biological molecule-containing biological compositions include, for example, serum, blood cell proteins, blood plasma concentrate, blood plasma protein fractions, purified or partially purified blood proteins or other components, a supernatant or a precipitate from any fractionation of the plasma, purified or partially purified blood components (e.g., proteins or lipids), mammalian colostrum, milk, urine, saliva, a cell lysate, cryoprecipitate, cryosupernatant, or portion or derivative thereof, compositions containing proteins induced in blood cells, and compositions containing products produced in cell culture by normal or transformed cells (e.g., via recombinant DNA or monoclonal antibody technology).
By xe2x80x9clipophilic moietyxe2x80x9d is meant a moiety selected from the list consisting of linear, branched, or cyclic saturated or unsaturated hydrocarbons with one to forty carbons, benzyl groups, or polycyclic aromatic groups.
By xe2x80x9creporter moietyxe2x80x9d is meant a UV adsorbing or fluorescent group which is added to the lipophilic quenching agent for the monitoring of removal of the lipophilic quenching agent and the quenched inactivating agent.
By xe2x80x9cviral inactivating conditionsxe2x80x9d is meant the conditions under which the viral particles are incubated with the selective ethyleneimine oligomer inactivating agents of this invention, including, for example, time of treatment, pH, temperature, salt composition and concentration of selective inactivating agent so as to inactivate the viral genome to the desired extent. Viral inactivating conditions are selected from the conditions for selective modification of nucleic acids described in U.S. patent application Ser. No. 08/855,378, hereby incorporated by reference.
The invention provides new methods for the quenching of viral inactivating agents and the subsequent removal of the quenching and inactivating agents from a biological composition. This method results in a biological composition that is relatively free not only of contaminating viruses, but also relatively free of quenched (i.e., non-reactive) inactivating agent and unreacted quenching agent. The invention provides methods which are compatible with methods of removing solvent and detergent from protein-containing preparations which are virally-inactivated by a solvent/detergent method.
Other features and advantages of the invention will be apparent from the following description and from the claims.